We have proposed a cross-bridge model of muscle contraction in which, during each cycle of ATP hydrolysis, the myosin cross-bridge alternates between a conformation in which the cross-bridge binds strongly to actin, and a conformation in which the cross-bridge binds weakly to actin with a rapid equilibrium existing between the attached and detached cross-bridge. While the cross-bridge is in the latter conformation the hydrolysis step and a separate rate limiting step occur. In the present study, we have tested several predictions of this model both in vitro and in single skinned rabbit muscle fibers. First, we measured 0-18 exchange with S-1 chemically cross-linked to actin and found that much less 0-18 exchange occurs than is predicted by our kinetic model. Yet, as predicted by our model, ATP hydrolysis does not appear to be the rate-limiting step in the ATPase cycle. Together these results suggest that the binding of actin to S-1 may affect the freedom of the rotation of Pi at the ative site of S-l. Since such an effect on 0-18 exchange has not previously been observed, it may have major implications for the mechanism of 0-18 exchange occurring both in vitro and in vivo in muscle and also in other systems such as mitochondria. We also studied the effect of AMPPNP and PPi on relaxation of force in single muscle fibers. Among other effects, our results suggested that AMPPNP and PPI show much weaker binding to actin bound cross-bridges than was previously observed by other workers. We therefore reinvestigated the binding of AMPPNP and PPI to acto.S-1 in vitro and here too we found much weaker binding than was previously reported. This result is important in interpreting the numerous structural and mechanical studies carried out with these ATP analogues on muscle fibers.